RTEF-1 variants and the use thereof for inhibition of angiogenesis

ABSTRACT

Dominant negative (DN) variants of transcriptional enhancer factor 1-related (RTEF-1) are described. DN RTEF-1 polypeptides may be directly targeted to cells or delivered in nucleic acid expression vectors to alter cellular transcription. Methods for inhibiting VEGF production and thereby treating angiogenic disorders such as cancer are described. For example, in certain aspects, DN RTEF-1 may be used to treat angiogenic disorders of the eye such as age related macular degeneration (AMD).

This application is a continuation of U.S. application Ser. No.14/269,806, filed May 5, 2014, which is a divisional of U.S. applicationSer. No. 12/134,626, filed Jun. 6, 2008, which claims priority to U.S.Provisional Application No. 60/942,249, filed Jun. 6, 2007. The entiretext of each of the above-referenced disclosures is specificallyincorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns the fields of molecular biology and specificallyconcerns processes involving blood vessel formation (angiogenesis).

2. Description of Related Art

Transcriptional enhancer factor 1-related (RTEF-1) gene is a member ofthe TEA DNA binding domain gene family. The TEA DNA binding domain genefamily is highly conserved from Aspergillus nidulans, yeast, Drosophila,mice to human. The TEA DNA binding family of proteins can be involved inboth activation and repression of different genes and their particularfunction can be modified by association with other proteins (Kaneko &DePamphilis, 1998). Expression of specific members of these genes hasbeen identified in various mammalian tissues, including heart, skeletalmuscle, pancreas, placenta, brain and lung (Stewart et al., 1996;Yasunami et al., 1996; Farrance et al., 1996). Isoforms arising fromalternative splicing of mRNA from a single gene, for transcriptionalenhancer factor-1 (TEF-1) have been identified within a single tissuesuch as the pancreas (Zuzarte et al., 2000; Jiang et al., 2000). Theexpression profile of these genes within the mammalian eye has not beenreported.

Transcripts of the RTEF-1 gene were first identified in chicken tissueand demonstrated to be enriched in cardiac and skeletal muscle (Farranceet al., 1996). The chicken RTEF-1 binds to the myocyte-specific CAT(M-CAT) cis DNA elements and regulates expression of muscle specificgenes, and requires muscle specific cofactors for full transcriptionalactivation. Random screening of 2166 human colorectal cancer cDNAlibrary identified a partial cDNA RTEF-1 sequence which lead to theisolation of a full length human homolog of the avian RTEF-1 from aheart cDNA library (Stewart et al., 1996; Frigerio et al., 1995).Northern blot analysis of human tissue indicated highest levels ofexpression in skeletal muscle and pancreas, with lower levels in heart,kidney and placenta, whereas message was not detected in liver, lung orbrain (Stewart et al., 1996). Northern blot analysis of the mousehomolog of RTEF-1 indicates a different tissue expression pattern whencompared to human. Adult mouse lung tissue expressed the highest level,with very low levels in kidney, heart and skeletal muscle andundetectable amounts in liver, thymus, spleen and brain, whereas RTEF-1message was abundant in mouse embryonic skeletal muscle (Yockey et al.,1996). An alternatively spliced mouse isoform of RTEF-1 that lacks exon5 when compare to the full length gene has been identified in mouseskeletal muscle cells (Yockey et al., 1996).

Vascular endothelial growth factor (VEGF) is one pro-angiogenic factorthat is known to be up regulated in retinal tissue under hypoxicconditions (Young et al., 1997; Pierce et al., 1996; Donahue et al.,1996; Pe'er et al., 1995). Recently the full length RTEF-1 protein hasbeen identified to not only bind to the VEGF promoter but also toup-regulate the expression of VEGF, for instance under hypoxicconditions in bovine aortic endothelial cells (BAEC) (Shie et al.,2004). Microarray analysis revealed that RTEF-1 expression wasup-regulated by 3-fold in BAEC under hypoxic conditions. Surprisingly,RTEF-1 mediated VEGF gene activation via interaction with Sp1 elementswithin the VEGF promoter and not M-CAT motifs. In addition RTEF mediatedexpression of VEGF is achieved independently of the hypoxia-induciblefactor (HIF-1) and hypoxia responsive element (HRE) pathway ofactivation (Shie et al., 2004).

VEGF over-expression has been implicated in a variety of angiogenicdisorders such as tumor angiogenesis and aberrant neovascularization.For example, it is well established that VEGF plays an important role inthe development and severity of retinopathy of prematurity (ROP) andother ocular neovascular diseases (Lashkari et al., 2000; Miller, 1997;Vannay et al., 2005; Young et al., 1997). Given the prominent role ofVEGF in such disorders a number of therapeutic strategies for inhibitingVEGF activity have been developed. However, current VEGF blockadetherapies typically involve inhibiting the interaction of extra cellularVEGF with cognate cell surface receptors. Thus, there is a need foralternative strategies for VEGF blockade such as method for inhibitingVEGF production.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides an isolated dominantnegative (DN) RTEF-1 polypeptide comprising an RTEF-1 amino acidsequence with one or more internal deletions. As used herein the termdominant negative means that the RTEF-1 variant suppresses or reducesthe activity of an intact RTEF-1 polypeptide as exemplified by SEQ IDNO:1. For example, in certain aspects, a DN RTEF-1 variant may bedefined as a polypeptide that when expressed in a cell inhibits orreduces VEGF promoter activity. Furthermore, in some cases, a DN RTEF-1may be defined as a polypeptide that reduces or inhibits hypoxia inducedor RTEF-1 (e.g., SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4) induced VEGFpromoter activity. In certain aspects, a RTEF-1 amino acid sequence maybe a mammalian RTEF-1 amino acid sequence, preferably a human RTEF-1amino acid sequence.

Thus, in certain aspects there is provided a DN RTEF-1 polypeptidecomprising one or more internal amino acid deletions. For example, incertain cases a DN RTEF-1 may comprise a deletion of amino acids encodedby exons 3, 4, 5, 6, 7, 8, 9 or 10. For example, in certain specificembodiments, a DN RTEF-1 may comprise a deletion of all of the aminoacids sequence encoded by exons 4, 5, 6, 7, 8 and/or 9. Furthermore, incertain specific cases, a DN RTEF-1 may comprise a partial deletion ofamino acids from exon 3 and/or exon 10, such as a deletion of about thelast 5 amino acids encoded by exon 3 or deletion of about the first 11amino acids encoded by exon 10. Furthermore it will be understood by theskilled artisan that a DN RTEF-1 may comprise amino acid substitutionsrelative to a wild type RTEF-1 sequence, such as human RTEF-1 sequence.Thus, in certain cases, a DN RTEF-1 may be defined as a RTEF-1polypeptide comprising one or more internal amino acid deletions whereinthe DN RTEF-1 is about or at least about 70, 75, 80, 85, 90, 95, 98 or99 percent identical to a wild type RTEF-1 sequence (e.g., SEQ ID NO:1,SEQ ID NO:2 or SEQ ID NO:4) over the undeleted amino acid region. Insome very specific aspects a RTEF-1 dominant negative polypeptide maycomprise a RTEF-1 amino acid sequence is about or at least about 70, 75,80, 85, 90, 95, 98 or 99 percent identical to SEQ ID NO:3 (the aminoacid sequence encoded by the 651 bp cDNA). In some very specific aspectsthe DN RTEF-1 polypeptide may comprise the sequence given by SEQ IDNO:3. Further embodiments of DN RTEF-1 polypeptides contemplated by theinvention are provided in the detailed description of the embodiments.

In some further aspects a DN RTEF-1 polypeptide may comprise a cellinternalization moiety. In some cases a cell internalization moiety maybe associated with or conjugated to a DN RTEF-1 polypeptide. Forexample, a DN RTEF-1 may be provided in complex with a liposomalvesicles thereby enabling the polypeptide to traverse the cell membrane.Furthermore, in some specific embodiments a cell internalization moietymay be a peptide, a polypeptide, an aptamer or an avimer (see forexample U.S. Applns. 20060234299 and 20060223114) sequence. For example,a cell internalization moiety may comprise amino acids from the HIV tat,HSV-1 tegument protein VP22, or Drosophila antennopedia. In certainfurther aspects, a cell internalization moiety may be a engineeredinternalization moiety such as the poly-Arginine, Methionine and Glycinepeptides described by Wright et al. (2003) and Rothbard et al. (2000).For example, a cell internalization moiety may be the RMRRMRRMRR (SEQ IDNO:23) exemplified herein. Thus, in some cases a polypeptide cellinternalization moiety and a DN RTEF-1 polypeptide may comprise a fusionprotein.

Thus, in certain cases, DN RTEF-1 fusion proteins are providedcomprising a cell internalization moiety and a DN RTEF-1 sequence. Theskilled artisan with understand that such fusion proteins mayadditionally comprises a one or more amino acid sequences separating thecell internalizing moiety and the DN RTEF-1 polypeptide sequence. Forexample, in some cases a linker sequence may separate these two domains.For example, a linker sequences may comprise a “flexible” amino acidswith a large number or degrees of conformational freedom such as a polyglycine linker. In some cases, a linker sequence may comprising aproteinase cleavage site. For instance, in certain aspects, a linkersequence may comprising a cleavage site that is recognized and cleavedby an intracellular proteinase thereby releasing a DN RTEF-1 sequencefrom the cell internalization sequence once the fusion protein has beeninternalized.

In further aspects of the invention a cell internalization moiety may befurther defined as a cell targeting moiety, that is a moiety that bindsto or is internalized by only a selected population of cells such ascells expressing a particular cellular receptor. Such a cell targetingmay, for example, comprise an antibody, a growth factor, a hormone, acytokine, an aptamer or an avimer that binds to a cell surface protein.As used herein the term antibody may refer to an IgA, IgM, IgE, IgG, aFab, a F(ab′)2, single chain antibody or paratope peptide. In certaincases, a cell targeting moiety of the invention may target a particulartype of cells such as a retinal, endothelial, iris or neuronal cell. Instill further aspects a cell targeting moiety of the invention may bedefined as cancer cell binding moiety. For example, in some veryspecific cases a cell targeting moiety of the invention may target acancer cell associated antigen such a gp240 or Her-2/neu.

In still further aspects of the invention a DN RTEF-1 polypeptide maycomprise additional amino acid sequences such as a cell traffickingsignals (e.g., cell secretion signal, a nuclear localization signal or anuclear export signal) or a reporter polypeptides such as an enzyme or afluorescence protein. In a preferred aspect for example, a DN RTEF-1polypeptide comprises a cellular secretion signal. For example, asexemplified herein a DN RTEF-1 polypeptide may comprise a secretionsequence from a human gene such as the IL-2 secretion signal sequence(MYRMQLLSCIALSLALVTNS, SEQ ID NO:22). Thus, in certain cases, a DNRTEF-1 polypeptide may comprise a cell internalization moiety and cellsecretion signal, thereby allowing the polypeptide to be secreted by onecells and internalized into a surrounding a cell.

In a further embodiment of the invention there is provided an isolatednucleic acid sequence comprising sequence encoding a DN RTEF-1polypeptide as described supra. Thus, a nucleic acid sequence encodingany of the DN RTEF-1 polypeptides or polypeptide fusion proteinsdescribed herein are also included as part of the instant invention. Theskilled artisan will understand that a variety of nucleic acid sequencemay be used to encode identical polypeptides in view of the degeneracyof genetic code. In certain cases for example the codon encoding anyparticular amino acid may be altered to improve cellular expression orto reduce the chance that a nucleic acid may recombine at a genomicRTEF-1 locus.

In preferred aspects, a nucleic acid sequence encoding a DN RTEF-1polypeptide is comprised in an expression cassette. As used herein theterm “expression cassette” means that additional nucleic acids sequencesare included that enable expression of DN RTEF-1 in a cell, or moreparticularly in a eukaryotic cell. Such additional sequences may, forexamples, comprise a promoter, an enhancer, intron sequences (e.g.,before after or with in the DN RTEF-1 coding region) or apolyadenylation signal sequence. The skilled artisan will recognize thatsequences included in an expression cassette may be used to alter theexpression characteristics of a DN RTEF-1. For instance, cell typespecific, conditional or inducible promoter sequences may be used torestrict DN RTEF-1 to selected cell types or growth conditions. Forexample, in certain cases a hypoxia inducible promoter may be used inRTEF-1 expression cassettes of the invention. Furthermore, in someinstances promoters with enhanced activity in cancer cells or cells ofthe eye may be employed. Furthermore, it is contemplated that certainalterations may be made to the RTEF-1 polypeptide sequence in order toenhance expression from an expression cassette for example, asexemplified herein, the initiation codon of a DN RTEF-1 may be changesto ATG to facilitate efficient translation.

In still further aspects of the invention a DN RTEF-1 coding sequencemay be comprised in an expression vector such as a viral expressionvector. Viral expression vectors for use according to the inventioninclude but are not limited to adenovirus, adeno-associated virus,herpes virus, SV-40, retrovirus and vaccinia virus vector systems. Incertain preferred aspects, a retroviral vector may be further defined asa lentiviral vector. In some cases such lentiviral vectors may beself-inactivating (SIN) lentiviral vector such as those described inU.S. Applns. 20030008374 and 20030082789 incorporated herein byreference.

In still further embodiments, the present invention concerns methods forreducing or inhibiting RTEF-1 dependent transcriptional activity. Asused herein the term RTEF-1 dependent transcriptional activity refers totranscription that is mediated or enhanced by expression of an fulllength or fully active RTEF-1 polypeptide, as exemplified by SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:4. Thus, in some respects, the inventionprovides methods for inhibiting or reducing VEGF promoter activity (andthereby VEGF expression) comprising expressing a DN RTEF-1 polypeptidein a cell. Thus, in a specific embodiment, there is provided a methodfor treating a patient with an angiogenic disorder comprisingadministering to the patient an effective amount of a therapeuticcomposition comprising a RTEF-1 dominant negative polypeptide or anucleic acid expression vector encoding a RTEF-1 dominant negativepolypeptide as described supra. In preferred aspects, methods describedherein may used to treat a human patient.

As used herein the term angiogenic disorder refers to disordersinvolving a undesirable vascularization such as ocularneovascularization, arterio-venous malformations, coronary restenosis,peripheral vessel restenosis, glomerulonephritis, rheumatoid arthritisor cancer (e.g., tumor vascularization). Thus, in certain cases, methodsof the invention may be used to treat ocular disorders such as maculardegeneration (e.g., age-related macular degeneration (AMD)), cornealgraft rejection, corneal neovascularization, retinopathy of prematurity(ROP) and diabetic retinopathy. For example, methods of the inventionmay be used in the treatment of wet or dry AMD. Thus, in certain cases,methods of the invention may be used to treat a number AMD associatedocular lesions such as predominantly classic, minimally classic, andoccult with no classic lesions (Gragoudas et al., 2004).

The skilled artisan will understand that additional antiangiogenictherapies may be used in combination or in conjunction with methods ofthe invention. Such additional therapies may be administered before,after or essentially simultaneously with the methods descried herein.For example additional antiangiogenic therapies may antagonize the VEGFand/or FGF signaling pathway. Thus, in some cases and additional therapymay comprise administration an antibody that binds to VEGF, a VEGFreceptor, FGF or an FGF receptor. In certain specific aspects, methodsand compositions of the invention may be used in conjunction withAVASTIN® (bevacizumab), LUCENTIS® (ranibizumab), MACUGEN® (pegaptanibsodium) or an anti-inflammatory drug. Thus, in certain specific casesthere is provided a therapeutic composition comprising a DN-RTEF-1composition and bevacizumab or pegaptanib sodium in a pharmaceuticallyacceptable carrier. In still further aspects a gene that regulatesangiogenesis may be delivered in conjunction with the methods of theinvention. For example, in some aspects, a gene that regulatesangiogenesis may be a tissue inhibitor of metalloproteinase, endostatin,angiostatin, endostatin XVIII, endostatin XV, kringle 1-5, PEX, theC-terminal hemopexin domain of matrix metalloproteinase-2, the kringle 5domain of human plasminogen, a fusion protein of endostatin andangiostatin, a fusion protein of endostatin and the kringle 5 domain ofhuman plasminogen, the monokine-induced by interferon-gamma (Mig), theinterferon-alpha inducible protein 10 (IP10), a fusion protein of Migand IP10, soluble FLT-1 (fins-like tyrosine kinase 1 receptor), andkinase insert domain receptor (KDR) gene. In certain specific aspects,such an angiogenic regulator gene may be delivered in a viral vectorsuch as the lentiviral vectors described in U.S. Pat. No. 7,122,181,incorporated herein by reference.

As described above, in certain aspects, the invention provides methodsfor treating cancer. Thus, in certain cases, described methods may beused to limit or reduce blood flow to a tumor thereby reducing tumorgrowth or metastasis. In certain cases, the methods herein may be usedto inhibit or treat metastatic cancers. A variety of cancer types may betreated with methods of the invention, for example a cancer fortreatment may be a bladder, blood, bone, bone marrow, brain, breast,colon, esophagus, eye, gastrointestinal, gum, head, kidney, liver, lung,nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, oruterus cancer. Furthermore additional anticancer therapies may be usedin combination or in conjunction with methods of the invention. Suchadditional therapies may be administered before, after or concomitantlywith methods of the invention. For example an additional anticancertherapy may be a chemotherapy, surgical therapy, an immunotherapy or aradiation therapy.

It is contemplated that DN RTEF-1 compositions of the invention may beadministered to a patient locally or systemically. For example, methodsof the invention may involve administering a DN RTEF-1 compositiontopically, intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intraocularly,intranasally, intravitreally, intravaginally, intrarectally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, by inhalation, by injection, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, via a catheter, or via a lavage. As described supra in somecases a DN RTEF-1 composition is delivered to the eye, administrationmay be, for example, via topical, subconjunctival, periocular,retrobulbar, subtenon, intracameral, intravitreal, intraocular,subretinal, posterior juxtascleral or suprachoroidal administration. Incertain aspects a DN RTEF-1 composition may be administered locally tothe eye by intraocular injection, topical administration (e.g., in aneye drop formulation).

In some further embodiments there is provided a pharmaceuticalcomposition of the invention comprised in a bottle said bottlecomprising an exit portal that enables drop-wise administration of thecomposition. In some cases, a pharmaceutical composition comprised in abottle comprises multiple doses however in certain aspects a bottlecomprises a single dose unit for administration to one or two eyes,preferable a single dose unit is comprised in 1-2 drops of theformulation. As used herein the term “bottle” refers to any fluidcontainer such as an ampoule, dropper or syringe.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1A-B: RTEF-1 mRNA splicing is altered during hypoxia. Retinal andiris endothelial cells were placed under hypoxic conditions and RTEF-1splicing was analyzed by RT-PCR. FIG. 1A, A schematic representation ofthe RTEF-1 splice variants identified by RT-PCR. Exon sequence for theuntranslated region (diagonal hatching) and amino acid coding region(open boxes) of each splice variant is shown. Putative RTEF-1 functionaldomains are also diagramed, checkered box indicates the TEA DNA bindingdomain (asterisks show position of predicted α-helices), solid boxindicates a nuclear localization signal (exon 4), vertical hatchingindicates an activation domain (proline rich domain (PRD)) andhorizontal hatching indicates the two STY domains in exons 9 and 10.FIG. 1B, A reproduction of agarose gel electrophoresis showing RTEF-1specific RT-PCR products prepared from primary cultures of human retinalvascular endothelial cells (RVEC). Lanes 1 & 4: DNA ladder; Lane 2: cDNAprepared from RVEC under normoxic conditions gave 2 products (1305 & 936bp in size); Lane 3: cDNA prepared from RVEC under hypoxic conditionsgave 3 products (1305, 936 & 447 bp in size).

FIG. 2: RTEF-1 variants activate or repress the VEGF promoter. 293Tcells were transfected with VEGF promoter reporter construct (VEGFpromoter driving secreted alkaline phosphotase) along with an expressionvector comprising the indicated RTEF-1 variant. Results show AP activityin the media was 6 hours post transfection with pSEAP-VEGF promoter pluspcDNA expression constructs comprising the 1305 bp (lane 1), 936 bp(lane 2), 651 bp (lane 3), ss-651-RMR by (lane 6) or 447 bp (lane 4)RTEF-1 variants. Lane five shows control VEGF promoter activity whencotransfected with an insert-less pcDNA control plasmid.

FIG. 3: RTEF-1 modulation of VEGF promoter activity is dependent on aportion of the VEGF promoter comprising 4 SP1 binding sites. Cells weretransfected as described above with pcDNA expression constructscomprising the 1305 bp (lane 1), 936 bp (lane 2), 651 bp (lane 3),ss-651 bp (lane 6) or 447 bp (lane 4) RTEF-1 variants and a reportervector comprising the intact VEGF promoter (solid boxes) or a VEGFpromoter with a deletion from nucleotide −113 to −57 (open boxes). Lanefive shows control VEGF promoter activity when cotransfected with aninsert-less pcDNA control plasmid.

FIG. 4: The 651 bp RTEF-1 acts as a dominant negative. Cells weretransfected as described above with pSEAP-VEGF and the 1305 bp (lanes 1,2), the 936 bp (lanes 3, 4) or the 447 bp (lane 5, 6) RTEF-1 varianteither alone (lanes 1, 3 and 5) or in addition to the ss-651-RMR byRTEF-1 variant (lanes 2, 4 and 6).

FIG. 5A-C: Detection of RTEF-1 polypeptides. Antibodies were raisedagainst an amino acid sequence unique to RTEF-1 but shared by each ofthe naturally occurring variants. FIG. 5A, is a amino acid alignmentbetween a region of RTEF-1 and related transcription factors. The aminoacid sequence used for antibody production is underlined. FIG. 5B, areproduction of an immunoblot using anti-RTEF-1 antibodies. Cellslysates used for analysis were from cell transfected with a pcDNA emptyvector (lanes 2 and 8) or a pcDNA expression vector for the 1305 bp(lanes 3, 9), 936 bp (lanes 4, 10), 651 bp (lanes 5, 11) or 447 bp(lanes 6, 11) RTEF-1 variant. Detection of the each RTEF-1 variant isindicated by the ellipses. Lanes 1 and 7 are molecular mass markers.Lanes 1-6 represent an overexposure of the image from lanes 7-12. FIG.5C, a reproduction of an immunoblot using anti-RTEF-1 antibodies todetect the RTEF-1 variant from cells transfected with a pcDNA 651 bpRTEF-1 expression vector. The expected ˜24 KDa polypeptide is indicatedby the arrow.

FIG. 6A-B: Expression of RTEF-1 variants in the eye. FIG. 6A, areproduction of an immunoblot showing RTEF-1 expression in normalprimate eye tissue. Immunoblot analysis was performed on protein fromretina (lane 1), choroid (lane 2) and iris (lane 3) tissue lysates. Mindicates a molecular mass markers. FIG. 6B, a reproduction of anethidium bromide stained agarose gel used to visualize semi-quantitativeRT-PCR productions generated using RTEF-1 specific primers. Lane 1 showsresults from CRAO retina RNA while lane 2 shows results from controlretinal RNA.

FIG. 7A-D: Immunohistochemistry analysis of RTEF-1 expression in primateeye tissue. FIG. 7A-B, Strong staining for RTEF-1 appears localized toiris (I), ciliary body (CB), optic nerve (ON) and retina (R). The cornea(C) and lens (L) were negative for RTEF-1 antibody hybridization. FIG.7C-D, The strongest RTEF-1 staining is in the ganglion cell layer (GCL)and the inner nuclear layer (INL). Staining appears to be localized toboth the cytoplasm and nucleus. Staining is scarce in the outer layers.

DETAILED DESCRIPTION OF THE INVENTION

Recently a number of strategies have been developed to inhibitangiogenic signaling for the purpose treating cancer and angiogenicdisorders such as AMD. In particular, a number of strategies havefocused on blockade of VEGF signaling by inhibiting the binding of VEGFwith one or both of its cell surface receptors. However, thesestrategies are unable to address the initial production of VEGF thatinitiates aberrant angiogenesis. Thus, new methods and compositions thatinhibit VEGF production may provide new methods for VEGF blockade andtreatments for resultant angiogenesis. To this end, in certain aspects,the instant invention provides a dominant negative transcription factorthat is integral in VEGF activation. Furthermore, since the instantinvention concerns the targeting of an intracellular processes,therapeutics of the invention may be targeted to specific cell typesthereby reducing undesirable systemic side effects. Thus, the instantinventions offers new methods to treat angiogenic disorders and/or waysto enhance the effectiveness of current VEGF blockade strategies.

RTEF-1 a member of a family of multifunctional transcription factors andhas been shown to be an activator of VEGF transcription, includinghypoxia induced VEGF transcription. However, as shown herein, multiplesplice variants of RTEF-1 are produced in cells and RTEF-1 polypeptidesproduced from alternative RNA splice variants comprise alteredtranscriptional function (FIG. 1A, B). It particular, one RTEF-1transcript of approximately 651 base pairs produces a polypeptide thatinhibits VEGF promoter activity (FIG. 2, compare lanes 3 and 5).Furthermore, this RTEF-1 variant was shown to be an even more effectiveinhibitor of the VEGF promoter when provided as a fusion protein with asecretion signal and cell internalization polypeptide FIG. 2 lane 6).Importantly, as shown in FIG. 4 the polypeptide from the 651 bp RTEF-1transcript acts in a dominant negative. That is the polypeptide not onlyreduces VEGF promoter activity but also blocks VEGF promoter enhancementby a other RTEF-1 protein isoforms. Furthermore, as shown in FIGS. 6 and7 RTEF is expressed in the tissues of the eye thereby implicating itsimportance in the development of ocular neovascular disorders. Studieshere indicate that RTEF-1 activation of VEGF production may for onefactor that contributes to the development of neovascular disorders.Thus, methods and compositions of the invention may provide a means forpreventing the early stages of neovascularization.

The instant invention provides the basis for new DN RTEF-1 polypeptidesand the use thereof to prevent or inhibit angiogenic disorders. DNRTEF-1 polypeptides may delivered directly to the intra cellular milieuor expressed in targeted cells to blockade VEGF production. Suchdominate negative polypeptides down regulate not only nascent VEGFproduction but also production of VEGF that is normally stimulated byRTEF-1 such bas during hypoxia. Thus, compositions of the invention, maybe used to reduce the ability of targeted cells and tissues to recruitnew blood vessel formation. This is of great interest in, for example,ocular neovascular disorders such as AMD where the invasion of bloodvessels is directly related to the pathogenesis of the disease.Furthermore, DN RTEF-1 may be used to treat tumors or tumor metastasesby reducing their ability to gain nutrients through new blood vesselformation. Thus, methods to slow tumor growth and/or induce tumorregression are also provided. Furthermore, since compositions of theinvention target intracellular transcription apprentice compositions ofthe invention may be used to target effected tissues by used of specificcell targeting/internalization moieties thereby reducing the sideeffects in other non-targeted tissues.

I. Dominate Negative RTEF-1 Polypeptides

A number of RTEF-1 variants are described and functionally characterizedherein. For example, the four sequence specifically studied herecomprise the following amino acid sequences.

SEQ ID NO: 1, encoded by a 1305 bp RTEF-1 human cDNA is a 434 amino acidprotein having the sequence:

LEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDGEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKDQAAKDKALQSMAAMSSAQIISATAFHSSMRLARGPGRPAVSGFWQGALPGQAETSHDVKPFSQQTYAQPPLPLPGFESPAGPAPSPSAPPAPPWQGRRRGSSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYLRPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE

SEQ ID NO: 2, encoded a 936 bp human cDNA is a 311 amino acid proteinhaving the sequence:

LEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDGEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKYNKHLFVHIGQSSPSYLRPYLEAVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHG AQHHIYRLVKE

SEQ ID NO: 3, encoded by a 651 bp human cDNA is a 216 amino acid proteinhaving the sequence:

LEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDGEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTSSFYGVSSQYESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVS ASEHGAQHHIYRLVKE

SEQ ID NO: 4, encoded by a 447 bp human cDNA is a 148 amino acid proteinhaving the sequence:

LEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDGEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVLARRKAREIQAKLKFWQGALPGQAETSHDVKPFSQHHIYRLVKE

As described supra, in certain aspects of the invention a dominantnegative (DN) RTEF-1 polypeptide may comprise one or more internal aminoacid deletions. For example, in some cases DN RTEF-1 may comprise adeletion of amino acids encoded by exons 3, 4, 5, 6, 7, 8, 9 or 10. Forexample, in certain aspects a DN-RTEF1 comprises the amino acid sequenceof SEQ ID NO:3 or a derivative thereof.

TABLE 1 RTEF-1 amino acid sequence by encoding exon ExonAmino acid sequence encoded  1 N/A  2LEGTAGTITSNEWSSPTSPEGSTASGGSQALDKPIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYG* (SEQ ID NO: 5)  3RNELIARYIKLRTGKTRTRKQ (SEQ ID NO: 6)  4 VSSHIQVLARRKAREIQAKLK(SEQ ID NO: 7)  5 DQAAKDKALQSMAAMSSAQIISATAFHSSMALARGPGRPAVSG(SEQ ID NO: 8)  6 FWQGALPGQAGTSHD* (SEQ ID NO: 9)  7VKPFSQQTYAVQPPLPLPG* (SEQ ID NO: 10)  8FESPAGPAPSPSAPPAPPWQGRSVASSKLWMLEFSAFLEQQQD PDT (SEQ ID NO: 11)  9YNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKDL FERGPSNAFFLVKFW(SEQ ID NO: 12) 10 ADLNTNIEDEGSSFYGVSSQYESPENMIITCSTKVCSFGKQVV EKVE(SEQ ID NO: 13) 11 TEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSV LENFTILQ(SEQ ID NO: 14) 12 VVTNRDTQETLLCIAYVFEVSASEHGAQHHIYRLVKE (SEQ ID NO: 15)*Indicates amino acid that are encoded by nucleic acid codons that aresplit between exons.

In additional aspects of the invention, DN RTEF-1 polypeptides may befurther modified by one or more amino substitutions while maintainingtheir transcriptional functions. For example, amino acid substitutionscan be made at one or more positions wherein the substitution is for anamino acid having a similar hydrophilicity. The importance of thehydropathic amino acid index in conferring interactive biologic functionon a protein is generally understood in the art (Kyte & Doolittle,1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Thus such conservative substitution can be madein an RETF-1 sequence and will likely only have mirror effects on theiractivity and ability to repress VEGF promoter activity. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (0.5); histidine −0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(2.3); phenylalanine (−2.5); tryptophan (−3.4). These values can be usedas a guide and thus substitution of amino acids whose hydrophilicityvalues are within ±2 are preferred, those that are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred. Thus, any of the DN RTEF-1 polypeptides described herein maybe modified by the substitution of an amino acid, for different, buthomologous amino acid with a similar hydrophilicity value. Amino acidswith hydrophilicities within +/−1.0, or +/−0.5 points are consideredhomologous.

II. Cell Internalization and Targeting Moieties

Cell internalization moieties for use herein may be any molecule incomplex (covalently or non-covalently) with a DN RTEF-1 that mediatetransport of the DN RTEF-1 across a cell membrane. Such internalizationmoieties may be peptides, polypeptides, hormones, growth factors,cytokines, aptamers or avimers. Furthermore, cell internalization moietymay mediate non-specific cell internalization or be a cell targetingmoiety that is internalized in a subpopulation of targeted cells.

For example, in certain embodiments, cell targeting moieties for use inthe current invention are antibodies. In general the term antibodyincludes, but is not limited to, polyclonal antibodies, monoclonalantibodies, single chain antibodies, humanized antibodies, minibodies,dibodies, tribodies as well as antibody fragments, such as Fab′, Fab,F(ab′)2, single domain antibodies and any mixture thereof. In some casesit is preferred that the cell targeting moiety is a single chainantibody (scFv). In a related embodiment, the cell targeting domain maybe an avimer polypeptide. Therefore, in certain cases the cell targetingconstructs of the invention are fusion proteins comprising a DN RTEF-1and a scFv or an avimer. In some very specific embodiments the celltargeting construct is a fusion protein comprising DN RTEF-1 polypeptidefused to a single chain antibody.

In certain aspects of the invention, a cell targeting moieties may be agrowth factor. For example, transforming growth factor, epidermal growthfactor, insulin-like growth factor, fibroblast growth factor, Blymphocyte stimulator (BLyS), heregulin, platelet-derived growth factor,vascular endothelial growth factor (VEGF), or hypoxia inducible factormay be used as a cell targeting moiety according to the invention. Thesegrowth factors enable the targeting of constructs to cells that expressthe cognate growth factor receptors. For example, VEGF can be used totarget cells that express FLK-1 and/or Flt-1. In still further aspectsthe cell targeting moiety may be a polypeptide BLyS (see U.S. Appln.20060171919).

In further aspects of the invention, a cell targeting moiety may be ahormone. Some examples of hormones for use in the invention include, butare not limited to, human chorionic gonadotropin, gonadotropin releasinghormone, an androgen, an estrogen, thyroid-stimulating hormone,follicle-stimulating hormone, luteinizing hormone, prolactin, growthhormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin,thyrotropin-releasing hormone, growth hormone releasing hormone,corticotropin-releasing hormone, somatostatin, dopamine, melatonin,thyroxine, calcitonin, parathyroid hormone, glucocorticoids,mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin,glucagon, amylin, erythropoitin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, leptin,thrombopoietin, angiotensinogen, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36.As discussed above targeting constructs that comprise a hormone enablemethod of targeting cell populations that comprise extracelluarreceptors for the indicated hormone.

In yet further embodiments of the invention, cell targeting moieties maybe cytokines. For example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9,IL10, IL11, IL12, IL13, IL14, IL15, IL-16, IL-17, IL-18,granulocyte-colony stimulating factor, macrophage-colony stimulatingfactor, granulocyte-macrophage colony stimulating factor, leukemiainhibitory factor, erythropoietin, granulocyte macrophage colonystimulating factor, oncostatin M, leukemia inhibitory factor, IFN-γ,IFN-α, IFN-β, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand,4-1BBL, TGF-β, IL 1α, IL-1□β, IL-1 RA, MIF and IGIF may all be used astargeting moieties according to the invention.

In certain aspects of the invention a cell targeting moiety of theinvention may be a cancer cell targeting moiety. It is well known thatcertain types of cancer cells aberrantly express surface molecules thatare unique as compared to surrounding tissue. Thus, cell targetingmoieties that bind to these surface molecules enable the targeteddelivery of DN RTEF-1 specifically to the cancers cells. For example, acell targeting moiety may bind to and be internalized by a lung, breast,brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck,esophageal, liver, skin, kidney, leukemia, bone, testicular, colon orbladder cancer cell. The skilled artisan will understand that theeffectiveness of cancer cell targeted DN RTEF-1 may, in some cases, becontingent upon the expression or expression level of a particularcancer marker on the cancer cell. Thus, in certain aspects there isprovided a method for treating a cancer with targeted DN RTEF-1comprising determining whether (or to what extent) the cancer cellexpresses a particular cell surface marker and administering DN RTEF-1targeted therapy (or another anticancer therapy) to the cancer cellsdepending on the expression level of a marker gene or polypeptide.

As discussed above cell targeting moieties according to the inventionmay be, for example, an antibody. For instance, a cell targeting moietyaccording the invention may bind to a skin cancer cell such as amelanoma cell. It has been demonstrated that the gp240 antigen isexpressed in variety of melanomas but not in normal tissues. Thus, incertain aspects of the invention, there is provided a cell targetingconstruct comprising an DN RTEF-1 and a cell targeting moiety that bindsto gp240. In some instances, the gp240 binding molecule may be anantibody, such as the ZME-018 (225.28S) antibody or the 9.2.27 antibody.In an even more preferred embodiment, the gp240 binding molecule may bea single chain antibody such as the scFvMEL antibody.

In yet further specific embodiments of the invention, cell targetingconstructs may be directed to breast cancer cells. For example celltargeting moieties that bind to Her-2/neu, such as anti-Her-2/neuantibodies may conjugated to a DN RTEF-1. One example of a such a celltargeting constructs are fusion proteins comprising the single chainanti-Her-2/neu antibody scFv23 and DN RTEF-1. Other scFv antibodies suchas scFv(FRP5) that bind to Her-2/neu may also be used in thecompositions and methods of the current invention (von Minckwitz et al.,2005).

In certain additional embodiments of the invention, it is envisionedthat cancer cell targeting moieties according to invention may have theability to bind to multiple types of cancer cells. For example, the 8H9monoclonal antibody and the single chain antibodies derived therefrombind to a glycoprotein that is expressed on breast cancers, sarcomas andneuroblastomas (Onda et al., 2004). Another example are the celltargeting agents described in U.S. Appln. 2004005647 and in Winthrop etal., 2003 that bind to MUC-1 an antigen that is expressed on a varietycancer types. Thus, it will be understood that in certain embodiments,cell targeting constructs according the invention may be targetedagainst a plurality of cancer or tumor types.

III. Methods for Producing Antibodies

The following methods exemplify some of the most common antibodyproduction methods.

A. Polyclonal Antibodies

Polyclonal antibodies generally are raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the antigen. Asused herein the term “antigen” refers to any polypeptide that will beused in the production of a antibodies. Antigens for use according tothe instant invention include in certain instances, cancer cell surfacemarker polypeptides and eye specific cell surface markers.

It may be useful to conjugate an antigen or a fragment containing thetarget amino acid sequence to a protein that is immunogenic in thespecies to be immunized, e.g. keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glytaraldehyde, succinic anhydride, SOCl₂, orR¹ N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg of 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to 1/10 the original amount of conjugate inFreud's complete adjuvant by subcutaneous injection at multiple sites. 7to 14 days later the animals are bled and the serum is assayed forspecific antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal boosted with the same antigen conjugate, butconjugated to a different protein and/or through a differentcross-linking reagent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response.

B. Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, monoclonal antibodies of the invention may be made usingthe hybridoma method first described by Kohler & Milstein (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding 1986).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the target antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (MA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods,Goding (1986). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison et al. (1984), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity for anyparticular antigen described herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for the targetantigen and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such ase.g., ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al. (1962); David et al. (1974); Pain et al. (1981); andNygren (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Zola, 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a purified target antigen or an immunologically reactiveportion thereof) to compete with the test sample analyte for bindingwith a limited amount of antibody. The amount of antigen in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies generally are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

C. Humanized Antibodies

As discussed previously, antibodies for use in the methods of theinvention may be polyclonal or monoclonal antibodies or fragmentsthereof. However, in some aspects it is preferred that the antibodiesare humanized such that they do not illict an immune response in subjectbeing treated. Methods for humanizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., 1986); Riechmann et al., 1988; Verhoeyenet al., 1988), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (Cabilly, supra), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties, forexample the ability bind to an be internalized by a target cell. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992,which is a continuation-in-part of application Ser. No. 07/715,272 filedJun. 14, 1991.

D. Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor(1984) and Brodeur et al. (1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.(1993); Jakobovits et al. (1993).

Alternatively, the phage display technology (McCafferty et al., 1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.

Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimicks some of theproperties of the B-cell. Phage display can be performed in a variety offormats; for their review see, e.g. Johnson et al. (1993). Severalsources of V-gene segments can be used for phage display. Clackson etal. (1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al. (1991), or Griffith et al. (1993).In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al., 1992).In this method, the affinity of “primary” human antibodies obtained byphage display can be improved by sequentially replacing the heavy andlight chain V region genes with repertoires of naturally occurringvariants (repertoires) of V domain genes obtained from unimmunizeddonors. This techniques allows the production of antibodies and antibodyfragments with affinities in the nM range. A strategy for making verylarge phage antibody repertoires (also known as “the mother-of-alllibraries”) has been described by Waterhouse et al. (1993), and theisolation of a high affinity human antibody directly from such largephage library has been reported. Gene shuffling can also be used toderive human antibodies from rodent antibodies, where the human antibodyhas similar affinities and specificities to the starting rodentantibody. According to this method, which is also referred to as“epitope imprinting”, the heavy or light chain V domain gene of rodentantibodies obtained by phage display technique is replaced with arepertoire of human V domain genes, creating rodent-human chimeras.Selection on antigen results in isolation of human variable capable ofrestoring a functional antigen-binding site, i.e. the epitope governs(imprints) the choice of partner. When the process is repeated in orderto replace the remaining rodent V domain, a human antibody is obtained(see PCT patent application WO 93/06213, published Apr. 1, 1993). Unliketraditional humanization of rodent antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no frameworkor CDR residues of rodent origin.

IV. Nucleic Acid Molecules

In certain aspects, the instant invention concerns nucleic acidmolecules encoding a DN RTEF-1 polypeptide. In certain aspects, a DNRTEF-1 nucleic acid sequence is comprised in a nucleic acid vector. Theterm “vector” is used to refer to a carrier nucleic acid molecule intowhich a nucleic acid sequence can be inserted for introduction into acell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

A. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the βlactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, world wide web at epd.isb-sib.ch/)could also be used to drive expression. Use of a T3, T7 or SP6cytoplasmic expression system is another possible embodiment. Eukaryoticcells can support cytoplasmic transcription from certain bacterialpromoters if the appropriate bacterial polymerase is provided, either aspart of the delivery complex or as an additional genetic expressionconstruct.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Nonlimiting examples of such regions include the human LIMK2 gene(Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et. al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et. al.,1999), human CD4 (Zhao-Emonet et. al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et. al., 1998), D1A dopamine receptor gene (Lee, et. al.,1997), insulin-like growth factor II (Wu et. al., 1997), and humanplatelet endothelial cell adhesion molecule-1 (Almendro et. al., 1996).

B. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

C. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et. al., 1999, Levensonet. al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

D. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et. al., 1997, herein incorporated by reference.)

E. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences. In certain specific cases apolyadenylation signal may be the signal from neuropilin-1 as describedin U.S. Appln. 20050175591.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

F. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

G. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

H. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et. al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

I. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). DN RTEF-1 components of the present invention may be aviral vector that encodes a DN RTEF-1 pollypeptide. Non-limitingexamples of virus vectors that may be used to deliver a nucleic acid ofthe present invention are described below.

1. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

2. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et. al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the delivery of DNRTEF-1 expression cassettes of the present invention as it has a highfrequency of integration and it can infect nondividing cells, thusmaking it useful for delivery of genes into mammalian cells, forexample, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broadhost range for infectivity (Tratschin et. al., 1984; Laughlin et. al.,1986; Lebkowski et. al., 1988; McLaughlin et. al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein byreference.

3. Retroviral Vectors

Retroviruses have promise as DN RTEF-1 delivery vectors in therapeuticsdue to their ability to integrate their genes into the host genome,transferring a large amount of foreign genetic material, infecting abroad spectrum of species and cell types and of being packaged inspecial cell lines (Miller, 1992).

In order to construct a DN RTEF-1 retroviral vector, a nucleic acid(e.g., one encoding a DN RTEF-1) is inserted into the viral genome inthe place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et. al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et. al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et. al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Methods for delivery of antiangiogenic moleculeswith lentiviral vectors have been previously described, see for exampleU.S. Pat. No. 7,122,181 incorporated herein by reference. Lentiviralvectors are well known in the art (see, for example, Naldini et. al.,1996; Zufferey et. al., 1997; Blomer et. al., 1997; U.S. Pat. Nos.6,013,516 and 5,994,136). Some examples of lentivirus include the HumanImmunodeficiency Viruses: HIV-1, HIV-2 and the Simian ImmunodeficiencyVirus: SIV. Lentiviral vectors have been generated by multiplyattenuating the HIV virulence genes, for example, the genes env, vif,vpr, vpu and nef are deleted making the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

4. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et. al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et.al., 1988; Horwich et. al., 1990).

5. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et. al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et. al., 1989).

J. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson et.al., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harland and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et. al.,1986; Potter et. al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et. al., 1990); byusing DEAE dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et. al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et. al., 1979; Nicolau et.al., 1987; Wong et. al., 1980; Kaneda et. al., 1989; Kato et. al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et. al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG mediatedtransformation of protoplasts (Omirulleh et. al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et. al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

V. Therapeutic Methods

A. Pharmaceutical Preparations

Therapeutic compositions for use in methods of the invention may beformulated into a pharmacologically acceptable format. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of an pharmaceuticalcomposition that contains at least one DN RTEF-1 polypeptide or nucleicacid active ingredient will be known to those of skill in the art inlight of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, incorporated herein by reference). A pharmaceuticallyacceptable carrier is preferably formulated for administration to ahuman, although in certain embodiments it may be desirable to use apharmaceutically acceptable carrier that is formulated foradministration to a non-human animal, such as a canine, but which wouldnot be acceptable (e.g., due to governmental regulations) foradministration to a human. Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The actual dosage amount of a composition of the present inventionadministered to a subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In particular embodiments, the compositions of the present invention aresuitable for application to mammalian eyes. For example, the formulationmay be a solution, a suspension, or a gel. In some embodiments, thecomposition is administered via a bioerodible implant, such as anintravitreal implant or an ocular insert, such as an ocular insertdesigned for placement against a conjunctival surface. In someembodiments, the therapeutic agent coats a medical device or implantabledevice.

In preferred aspects the formulation of the invention will be applied tothe eye in aqueous solution in the form of drops. These drops may bedelivered from a single dose ampoule which may preferably be sterile andthus rendering bacteriostatic components of the formulation unnecessary.Alternatively, the drops may be delivered from a multi-dose bottle whichmay preferably comprise a device which extracts preservative from theformulation as it is delivered, such devices being known in the art.

In other aspects, components of the invention may be delivered to theeye as a concentrated gel or similar vehicle which forms dissolvableinserts that are placed beneath the eyelids.

Furthermore, the therapeutic compositions of the present invention maybe administered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic composition is determined basedon the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.Thus, in some case dosages can be determined by measuring for examplechanges in serum insulin or glucose levels of a subject.

Precise amounts of the therapeutic composition may also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting the dose include the physical and clinical state ofthe patient, the route of administration, the intended goal of treatment(e.g., alleviation of symptoms versus attaining a particular seruminsulin or glucose concentration) and the potency, stability andtoxicity of the particular therapeutic substance.

In particular embodiments, the compositions of the present invention aresuitable for application to mammalian eyes. For example, the formulationmay be a solution, a suspension, or a gel. In some embodiments, thecomposition is administered via a bioerodible implant, such as anintravitreal implant or an ocular insert, such as an ocular insertdesigned for placement against a conjunctival surface. In someembodiments, the therapeutic agent coats a medical device or implantabledevice.

In preferred aspects the formulation of the invention will be applied tothe eye in aqueous solution in the form of drops. These drops may bedelivered from a single dose ampoule which may preferably be sterile andthus rendering bacteriostatic components of the formulation unnecessary.Alternatively, the drops may be delivered from a multi-dose bottle whichmay preferably comprise a device which extracts preservative from theformulation as it is delivered, such devices being known in the art.

In other aspects, components of the invention may be delivered to theeye as a concentrated gel or similar vehicle which forms dissolvableinserts that are placed beneath the eyelids.

B. Additional Therapies

As discussed supra in certain aspects therapeutic methods of theinvention may be used in combination or in conjunction with additionalantiangiogenic or anticancer therapies.

1. Chemotherapy

In certain embodiments of the invention DN RTEF-1 is administered inconjunction with a chemo therapeutic agent. For example, cisplatin(CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, paclitaxel, gemcitabien, navelbine,farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil,vincristin, Velcade, vinblastin and methotrexate, or any analog orderivative variant of the foregoing may used in methods according to theinvention.

2. Radiotherapy

In certain further embodiments of the invention DN RTEF-1 compositionsmay be used to sensitize cell to radiation therapy. Radio therapy mayinclude, for example, γ-rays, X-rays, and/or the directed delivery ofradioisotopes to tumor cells. In certain instances microwaves and/orUV-irradiation may also used according to methods of the invention.Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 wk), to single doses of 2000 to6510 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radio therapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

3. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B, Her-2/neu, gp240 and p155.

4. Genes

In yet another embodiment, gene therapy in which a therapeuticpolynucleotide is administered before, after, or at the same time as acell targeting construct of the present invention. Delivery of DN RTEF-1in conjunction with a vector encoding one or more additional geneproducts may have a combined anti-hyperproliferative effect on targettissues. A variety of genes are encompassed within the invention, forexample a gene encoding p53 may be delivered in conjunction with DNRTEF-1 compositions.

5. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. A DN RTEF-1 therapyof the invention may be employed alone or in combination with acytotoxic therapy as neoadjuvant surgical therapy, such as to reducetumor size prior to resection, or it may be employed as postadjuvantsurgical therapy, such as to sterilize a surgical bed following removalof part or all of a tumor.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

6. Other agents

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

EXAMPLES

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques and/or compositions discovered by the inventor tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Experimental Methods

Primary Ocular Vascular Endothelial Cell Isolation and Culture

All use of human cells and tissue was in accordance with approvedinstitutional review board protocols. Primary cultures of endothelialcells isolated from human retina were established using establishedprotocols and used as a source for mRNA (Kanda et al., 1998; Silvermanet al., 2005). Human cadaver eyes were obtained from anonymous donors(Lion's Eye Bank, Portland, Oreg.) within 24 hours of death. Donors hadno history of cardiovascular or ocular disease and ranged in age from16-42. Briefly, these retinal and iris tissues were asepticallydissected and separated away from donor eyes, and digested in 0.2%collagenase (Sigma Chemical Co, St Louis, Mo.) and endothelial cells(EC) were isolated from other cell types by using mouse monoclonalanti-human CD31 antibody-coated magnetic beads (Dynal Biotech, Inc.,Lake Success, N.Y.). ECs were cultured in complete MCDB-131 medium(Clonetics/BioWhittaker, Walkersville, Md.) supplemented with 10% fetalbovine serum and antibiotics. Cells were used at passages 2 to 5. After2 rounds of magnetic bead separation the EC cultures were more than99.5% pure, as evaluated by morphologic criteria, expression of CD31 andvon Willebrand factor, and uptake of acetylated low density lipoprotein(Silverman et al., 2005).

Induction of Hypoxia.

Retinal and iris endothelial cells were cultured to 80% confluence in 60mm diameter culture dishes and then placed in an air tight ModulatorIncubator Chamber (Billups-Rothenberg, Del Mar, Calif.). A 1% O₂, 5% CO₂and remainder N2 gas mix was flushed through the chamber for exactly 5minutes whereupon the chamber was sealed and placed into a humidified37° C. incubator. After 8 hours the chamber was flushed again for 5minutes with the hypoxic gas mix and sealed and incubated for a further8 hours and then flushed again and incubated for another 8 hours atwhich point total RNA was isolated.

Total RNA Extraction and RT-PCR

Total RNA was isolated using an RNAqueous kit (Ambion Inc, Austin, Tex.)according to manufacturer's protocol and 50 ng of this RNA was used withan oligo-dT primer first strand synthesis (SuperScript II, Stratagene,La Jolla, Calif.). The following primers, F1: 5′-ttggagggcacggccggca-3′(SEQ ID N0:20) and R1: 5′-tcattctttcaccagcctgta-3′ (SEQ ID N0:21)designed from published RTEF-1 sequence (NCBI accession # U63824) wasused for second strand PCR amplification using standard conditions.Amplified products were subjected to electrophoresis and visualized in a1.5% agarose gel and subsequently purified from the gel (Qiaquick GelExtraction, Qiagen, Valencia, Calif.) for standard dideoxynucleotidesequencing on an Abi 310 automated sequencer.

Reporter Gene Analysis

Full length RTEF-1 isoforms were directionally cloned into the pcDNA 3.1expression plasmid (Invitrogen, Carlsbad, Calif.). The predicted TTGstart was converted to ATG within the forward primer sequence. HumanVEGF 5′ proximal promoter fragment of 1,136 bp containing 54 bp of 5′UTRand 1,082 bp upstream of the transcription start site was directionallycloned 5′ to the secretable alkaline phosphatase (SEAP) gene within thepSEAP reporter plasmid (Clontech, Mountain View, Calif.). Promoterfragments with deletions were constructed by first amplifying the 5′ endof the promoter and 3′ end of the promoter and subsequent ligation ofthe amplified products. The ligated products lacking the region ofinterest were then amplified and directionally cloned into thepromoterless pSEAP vector. All constructs were sequenced on both strandsfor verification prior to transfection studies.

Transfection Assays.

Transfection was performed using the Amaxa Nucleofection Device (AmaxaInc, Gaithersburg, Md.), Amaxa reagents and standard manufacturer'sprotocol. Briefly, 293T cells were cultured in 10% DMEM media till 80%confluent, trypsinized and collected. One million cells was used pernucleofection. One million cells were resuspended in 100 ul ofNucleofect solution and 5 μl (containing 2 μg) of total plasmid DNA,electroporated (program #A023 on Nucleofection Device) and thenimmediately resuspended in 1 ml of prewarmed media and seeded into asingle well of a 6 well plate. Cells were allowed to recover for 16-18hours and the media was carefully removed and replaced with exactly 500ml of fresh media. After exactly 6 hours of incubation 150 μl of mediawas carefully removed and 25 μl of this was either assayed immediatelyor stored at −20° C. for future SEAP analysis. Three separate 25 μlmedia aliquots were used for SEAP analysis according to manufacturer'sprotocol (BD Biosciences, San Jose, Calif.) and the SEAP value for all 3readings were averaged for comparison to triplicate repeat experiments.

Each cotransfection was repeated at least 3 times in a single experimentand each experiment repeated again independently 2 more times withseparate plasmid preparations (n=9-12). One representative experiment ispresented in figures. Statistical analysis was performed using aStudent's t-test (two-tailed) to compare the 3 or 4 samples in a singleexperiment. Bonferroni correction for multiple testing was applied and aP<0.01 was considered as significant.

For each cotransfection assay (when 2 plasmids were transfected togetherin the same tube) the copy number of each plasmid was adjusted to beequivalent to the copy number of the largest plasmid used. The pSEAPvector without a promoter and the pcDNA 3.1 expression plasmid with noinsert served as negative controls. For each nucleofection experiment 2separate positive control plasmids, a SV40 promoter pSEAP plasmid and apGFPmax vector, were transfected at the same time to ensure efficientand equal transfection efficiencies. The pSEAP plasmid with an SV40promoter served as a positive control for subsequent SEAP proteinanalysis. The pGFPmax vector also served as positive control fortransfection for each batch of cells allowing visual confirmation ofconsistent transfection efficiency. Nucleofection consistently gave80-90% transfection efficiency in 293T cells in all experiments.

Example 2 Novel Isoforms of RTEF-1 Exist within Hypoxic and NormalOcular Vascular Endothelial Cells

Amplification from cDNA prepared from primary cultures of human retinal(PRVEC) and iris (PIVEC) vascular endothelial cells, using the F1 and R1primer pair, gave products of approximately 1305 bp and 936 bp (FIG.1B). Using the same primer pair amplification from cDNA isolated fromPRVEC that had been cultured under hypoxic conditions for 24 hours,prior to isolation of mRNA, gave an additional product of approximately447 bp (FIG. 1B). The 651 bp cDNA was isolated from Human primaryretinal vascular endothelial cells (PRVEC).

Sequencing analysis revealed that the largest product was identical tothe full length 1305 bp RTEF-1 gene spanning from the start to the stopcodon (SEQ ID NO:1), whereas the 936 bp, 651 bp and 447 bp transcriptswere alternate spliced transcripts of the 1305 bp product. The followingdescription of codons will be numbered according to the sequence in the1305 bp transcript which consists of 435 codons with the proteininitiating codon being 1 and the stop codon being 435. Exons 5 to 8,four of the eleven exons that are predicted to code for the proteinportion of the 1305 bp transcript, are spliced out of the 936 bp version(FIG. 1A). Not only is exon 5 lacking in the 447 bp isoform, but anunusual in frame splice event occurs in the middle of exon 7 whichsplices out from Gln-83 in exon 7 to codon Gln-425 within exon 12 (FIG.1A). In the case of the 651 bp isoform a 5′ portion of exon 3 is spliceddirectly into an internal splice acceptor site in exon 10 therebycompletely removing exons 4, 5, 6, 7, 8 and 9 from the transcript.

The 1305 bp product shows identity to the transcriptional enhancerfactor-1 related (RTEF-1) gene originally identified in human cardiac,skeletal muscle, pancreas and lung tissue (Stewart et al., 1996). Twoother RTEF isoforms, variant 2 (accession #NM 201441) which lack exon 5from Asp-119 to Gly-161 and variant 3 (accession #NM 201443) whichemploys a downstream protein initiation site at Met-130, have previouslybeen reported. The 936 bp, 651 bp and 447 bp isoforms identified withinhuman ocular vascular cells have not been identified in any other humantissue to date.

The full length 1305 bp transcript encodes a polypeptide having 434amino acids with a predicted molecule weight of ˜48.6 KDa. Thispolypeptide comprises 50 strongly basic (K,R), 47 strongly acidic (D,E),133 hydrophobic (A, I, L, F, W, V) and 124 polar (N, C, Q, S, T, Y)amino acids. The predicted isoelectric point is 8.248 and the predictedcharge is 4.799 at pH 7.0. Each of the identified RTEF-1 isoforms appearto utilize a non-canonical TTG (UUG) start codon resulting in an aminoterminal lysine residue.

The 936 bp transcript encodes a polypeptide having 311 amino acids witha predicted molecule weight of ˜35.6 KDa. This polypeptide comprises 40strongly basic (K,R), 38 strongly acidic (D,E), 93 hydrophobic (A, I, L,F, W, V) and 92 polar (N, C, Q, S, T, Y) amino acids. The predictedisoelectric point is 8.037 and the predicted charge is 3.458 at pH 7.0.

The 651 bp transcript encodes a polypeptide having 216 amino acids witha predicted molecule weight of ˜24.4 KDa. This polypeptide comprises 22strongly basic (K,R), 27 strongly acidic (D,E), 60 hydrophobic (A, I, L,F, W, V) and 71 polar (N, C, Q, S, T, Y) amino acids. The predictedisoelectric point is 6.039 and the predicted charge is −4.046 at pH 7.0.The 651 bp isoform is spliced in frame from within exon 3 after Thr-92into the middle of exon 10 at Ser-311 (all numbering based in thelargest RTEF-1 isoform (SEQID NO:1)). Thus, the 651 bp isoform containsall of exon 2, most of exon 3 (lacks 5 of the 22 amino acids in exon 3),most of exon 10 (lacks the first 11 amino acids of exon 10) and completeexons 11 and 12. This results in retention of most of the TEA bindingdomain but with loss of one of the 3 predicted α-helices and theputative nuclear localization signal (Leu-105 to Lys-109) normallycontained within the 72 amino acid TEA domain. The Proline Rich Domain(PRD), activation domain and the first STY domain (Ser-253 to Ser-271)is also lacking in the 651 bp isoform. Interestingly, the splice eventinto exon 10 starts at Ser-11 within this exon (i.e., the 11^(th) aminoacid in exon 10) which is the very start of the second STY domain(Ser-253 to Ser-336). This splice event results in fusion of a partialTEA domain, lacking a putative nuclear localization signal, directlywith a STY domain.

The 447 bp transcript encodes a polypeptide having 148 amino acids witha predicted molecule weight of ˜16.5 KDa. This polypeptide comprises 22strongly basic (K,R), 17 strongly acidic (D,E), 43 hydrophobic (A, I, L,F, W, V) and 40 polar (N, C, Q, S, T, Y) amino acids. The predictedisoelectric point is 9.444 and the predicted charge is 5.561 at pH 7.0.

The predicted protein sequence for both the 936 bp and 447 bp isoformscontain the 72 amino acid TEA domain (Asp-38 to Lys-109) which contains3 predicted α-helices and a putative nuclear localization signal(Leu-105 to Lys-109). However within the C-terminal domain a prolinerich-domain (Pro-189 to Pro-213) spanning the last 6 amino acids of exon7 and the first 19 residues of exon 8 is missing from the 447 bp isoform(FIG. 1A). In addition two STY domains (Ser-253 to Ser-271 and Ser-311to Ser-336), a region rich with hydroxylated residues such as serine,threonine and tyrosine, one located within exon 9 and the other withinexon 10 are also lacking in the 447 bp isoform (FIG. 1A).

Example 3 The Effects of Novel RTEF-1 Isoforms on Expression from theVEGF Promoter

It has been shown that the polypeptide resulting from the 1305 bpisoform acts as a transcriptional stimulator of VEGF, in bovine aorticendothelial cells, via binding to a Sp1 site (Shie et al., 2004). Thus,studies were conducted to investigate whether the new isoforms were alsocapable of stimulating expression from the human VEGF promoter. The 5′proximal promoter of the human VEGF gene, consisting of 54 bp of 5′UTRand 1,082 bp upstream of the transcription initiation site was clonedinto a pSEAP reporter plasmid and the RTEF-1 isoforms were cloned into apcDNA expression vector. Due to difficulties in nucleotransfection ofplasmid DNA into primary cultures of ocular vascular endothelial cells,293T cells were used as a substitute cell line for transfection studies.Co-transfection of the VEGF promoter-reporter plasmid with one of eachof the RTEF-1 isoforms indicate that the 1305 bp, 936 bp and 447 bpisoforms up-regulated expression of the reporter from the VEGF promoter(lanes 1, 2, and 4 FIG. 2). However, interestingly, the 651 bp isoformdown regulated expression from the VEGF promoter (lane 3, FIG. 2). Thefull length 1305 bp RTEF-1 product and the 936 bp isoform enhancedexpression between 3-4 fold significantly higher than background(P=0.001), and no difference was observed between these 2 isoforms(P=0.01) after correcting for multiple testing. The 447 bp isoformstimulated expression about 10-15 fold (average 12×) above backgroundexpression (P=0.0003). Each co-transfection experiment was repeated intriplicate on three separate occasions with the same results.

The 651 bp isoform (lane 3, FIG. 2) significantly down-regulatedexpression (P=0.0026) from the VEGF promoter relative to the control(Lane 5, FIG. 2). Surprisingly, the modified version of the 651 bpisoform, the SS-651 bp-RMR product described below (Lane 6, FIG. 2) notonly suppressed expression from the VEGF promoter relative to thecontrol (P=0.0009) but was even more potent at inhibiting expressionthan the 651 bp isoform (P=0.0008). The 651 bp isoform (lane 3, FIG. 2)inhibited expression approximately 3-fold lower than expression observedin the control, whereas the SS-651 bp-RMR version (lane 6, FIG. 2)inhibited expression about 10-fold lower than then control (shown inlane 5, FIG. 2). The potency of the SS-651 bp-RMR is likely due to thefact that this molecule is secreted out of the cell of productioncombined with its ability to be imported into neighboring cells.

The ss-651-RMR by RTEF-1 comprises the coding region for the 651 bpisoform of RTEF-1 fused at the N-terminus to the human IL-2 secretionsignal sequence (SEQ ID NO:22) and fused at the C-terminus to theinternalization moiety (SEQ ID NO:23). This created the “ss-651-RMR”product, which is secretable from expressing cells and importable intosurrounding cells.

Example 4 Sp1 Elements are Required for Maximal VEGF Promoter Activitybut are not Essential for RTEF Enhancer Activity

Prior studies demonstrated that the full length RTEF-1 isoform binds toand requires a Sp1 element for it to augment VEGF promoter activity. Ina previous study mutation of this Sp1 site situated at −97 to −89 bpresulted in loss of RTEF-1 enhancer activity (Shie et al., 2004). In thesame study three other Sp1 sites within the same region −86 to −58 bpwere found not to be essential for RTEF-1 enhancer activity. To testwhether the new RTEF-1 isoforms required Sp1 sites for enhanceractivity, the VEGF promoter with all four Sp1 sites deleted, from −113bp to −58 bp, was cloned into a pSEAP vector and was co-transfected witheach isoform. A comparison of background reporter gene expression fromthe full length and the Sp1-negative VEGF promoter indicates that lossof Sp1 elements results in a dramatic 30 fold decrease in reporterexpression (FIG. 3). This would suggest that at least one of the fourSp1 elements within the proximal promoter is essential for enhancingoverall expression. A similar level of depressed expression was noted inco-transfection experiments with the Sp1-negative and each isoform (FIG.3, compare filled bars with open bars). However, the same trend ofenhancement was still observed with the cotransfection experiments witheach of the isoforms for the Sp1-negative promoter assay (FIG. 3). A3-fold, 4-fold and 12-fold enhancement above background for the 1305 bp,936 bp and the 447 bp isoforms, respectively. Thus, the level ofenhancement above background afforded by each isoform is the sameregardless of whether Sp1 elements are present within the VEGF promoter.

The 651 bp isoform remains able to inhibit expression relative to thecontrol in the absence of the Sp1 sites within the VEGF promoter. Thus,regulation of VEGF promoter is the same for each isoform is the sameregardless of whether Sp1 elements are present or not. The 651 bpfragment is capable of inhibiting the enhancer effect of the 1305 bp,936 bp and 447 bp isoforms from the VEGF promoter in a competitivemanner. That is, introducing increased amounts of 651 bp in conjunctionwith any of the other enhancing isoforms that normally upregulate theexpression from the VEGF promoter resulted in competitive inhibition ofthe enhancer activity.

Example 5 Dominant Negative Transcriptional Activity of the Polypeptidefrom the 651 bp RTEF-1 cDNA

To investigate the effects of the 651 bp isoform on the VEGF promoterenhancement of other RTEF-1 isoforms further 239T transfectionexperiments were undertaken. Briefly, cells were transfected with theindicated RTEF-1 enhancer expression construct (i.e., 1305 bp, 936 bp or447 bp), a VEGF reporter vector and either an expression plasmid for the651 bp RTEF-1 isoform or an empty vector control. Following transfectionVEGF promoter activity was accessed by reporter gene assay as describedpreviously. Results of these studies are shown in FIG. 4. In each casethe enhancement activity of RTEF-1 isoforms (1305 bp, 936 bp and 447 bp)on VEGF promoter activity was repressed by co expression of the RTEF-1651 bp isoform.

Example 6 RTEF-1 cDNA Expression Vectors Produce Expected Polypeptidesin Cells

To confirm the expression of the indicated RTEF-1 polypeptides in cells293T cells were transfected with the a control (empty vector) pcDNAexpression vector or an expression vector for the 1305 bp, 936 bp, 651bp or 447 bp cDNA sequences. Following transfection cell lysates wereanalyzed by Western blot using an anti-RTEF-1 antisera. Anti-RTEF-1antisera was raised against an RTEF-1 peptide corresponding to aminoacids 2-14 of the full length sequence. Antibodies for Western blot weredirected to an RTEF-1 epitope that was unique relative to related humanTEA proteins but shared by each of the RTEF-1 isoforms that weretransfected (FIG. 5A). Results of the studies (FIG. 5B) demonstrate thateach of the expected RTEF-1 polypeptides was expressed in transfectedcells though in the case of the polypeptide from the 651 bp cDNAexpression levels were quite low.

Example 7 In Vivo Expression of RTEF-1 in Eye Tissue

The expression of RTEF-1 isoforms in normal primate eye tissue wasfurther studied. Western blot analysis with an anti-RTEF-1 antibody thatbinds to each protein isoform demonstrated RTEF-1 expression in certaintissues of the eye. Expression of RTEF-1 appeared highest in choroid andlowest in retina (FIG. 6A). Detected protein products that migratedslower than the 75 kD mass marker seem to be the full length RTEF-1(1305 bp) isoform (FIG. 6A, upper panel). Products migrating between the23 and 25 kD mass markers are believed to arise from the 651 bp isoform(FIG. 6A, lower panel).

In further studies that expression of RTEF-1 isoforms in the CRAO modelwere studied by RT-PCR. Results showed that full length (1305 bp) RTEF-1RNA was preferentially expressed in CRAO retina relative to controlretinal tissue (FIG. 6B, compare lanes 1 and 2).

To further assess the cellular distribution of RTEF-1 expression in eyetissues eye tissues were analyzed by immunohistochemistry with an RTEF-1binding antibody. Results demonstrated the expression of RTEF-1 in theiris, ciliary body, optic nerve and retina (FIG. 7A-B).

Example 8 Localization of the RTEF-1 Isoforms

The RTEF-1 isoforms (1305 bp, 651 bp, and 447 bp) were cloned into apMAX-FP-N vector (Amaxa Inc, Gaithersburg, Md.) with a differentfluorescent protein fused to the carboxyl end of each of the isoformsrespectively. The 1305 bp isoform was fused with a green fluorescentprotein (GFP), while 651 bp isoform was fused with a red fluorescentprotein (RFP) and the 447 bp isoform was fused with a yellow fluorescentprotein (YFP). The 651 bp isoform was also cloned into a pHR-CMV-eGFPvector with a hIL-2 secretion signal and a RMR transport motif toproduce a ss-651-RMR RTEF-1-GFP fusion protein. Each construct wasverified by sequencing analysis. Human 293T cells were plated into 6well plates at a density of 3×10⁵ cells per well. Cells were grown to80% confluency in DMEM media supplemented with 10% FBS and 1×concentration of penicillin-streptomycin-amphotericin. Each constructwas transfected into the treated 293T cells via electroporation usingthe Amaxa Nucleofecter II apparatus (Amaxa Inc, Gaithersburg, Md.).Cells were incubated for 24 hours at 37° C., with 5% of CO₂. Alltransfection reactions were observed using fluorescent microscopy forfluorescent activity, and were photo-documented to record localizationpatterns of the RTEF-1 protein isoforms.

The fluorescent microscopy analysis demonstrates that the twoVEGF-enhancer RTEF-1 isoforms (1305 bp and 447 bp) containing a nuclearlocalization signal were found localized to the nucleus of the cell.Furthermore, the inhibitory isoform 651 was found concentrated in thecytoplasm, outside and surrounding the nucleus. However, with a hiL-2secretion signal sequence and a RMR transport motif, the ss-651-RMRRTEF-1 isoform was found localizing in the cell nucleus.

To confirm the localization pattern of the RTEF-1 isoforms, westernimmunoblot analysis was performed for the each cellular fraction withcells transfected with RTEF-1 isoforms. For these studies, cells weretransfected with 2 μg of RTEF-1 isoforms and grown 24 hours. Media wasthen changed to serum-free DMEM and grown for an additional 48 hours.Cellular fractions were isolated and collected. Media was treated withTCA to precipitate all residual proteins in the media. RTEF-1 specificantibody, which does not distinguish among the three isoforms, was usedat a concentration of 1/5000 to detect the presence of RTEF-1 protein.The nuclear fractions, cytoplasmic fractions and media were analyzedwith the samples from cells transfected with a pcDNA empty vector, or apcDNA expression vector for the 1305 bp, 936 bp, 651 bp, ss-651-RMR, or447 bp RTEF-1 variant. The results indicated that the VEGF enhancerisoforms 1305 bp and 447 bp, increase the expression of VEGF via bindingto the chromosome DNA. However, it is surprising that the 651 bp RTEF-1negative dominant isoform localizes in the cytoplasm even though it cancompetitively inhibit the action of the enhancer RTEF-1 isoforms whichlocalize in the nucleus.

Example 9 RTEF-1 Protein is Present within Human Ocular Melanoma Cells

VEGF is a key protein responsible for the development of various ocularneovascular diseases and establishment of ocular tumors. Identificationof proteins that regulate the expression of the VEGF gene will help tounderstand the etiology and progression of ocular tumors. The mostcommon intraocular cancer in adults is ocular melanoma (OM) and can leadto local tissue damage, loss of vision and has a tendency to metastasizewhich has significant consequences on patient morbidity and mortality.

The various human RTEF-1 isoforms are able to differentially potentiateexpression from the VEGF 5′ proximal promoter region. Since upregulationof VEGF mediates angiogenesis, inflammation and tumor progression, theinventors believe that RTEF-1 may play a role in the development andadvancement of vascularized human ocular tumors such as melanoma, andpossibly other non-ocular tumors. The inventors investigated whetherRTEF-1 protein is present within human ocular melanoma cells byimmunohistochemistry methods. A section of a human eye which had amelanoma tumor was stained with an antibody that recognizes the humanRTEF-1 protein (but which does not distinguish as among the threeisoforms). A slide containing the section was examined using microscopy.The results demonstrated that RTEF-1 protein (observed as red staining)is present within human ocular melanoma cell. The melanoma cells lookbrown, due to the presence of melanin pigment. The cells stained red andbrown are tumor cells with RTEF-1 protein. The high level of RTEF-1 inthose tumor cells suggests that the RTEF-1 may upregulate the VEGF genewithin these cells, and promote cell proliferation and tumor expansion.

Thus, using the 651 bp RTEF-1 isoform may be used to repress VEGFexpression in these melanoma cells and to inhibit the growth of thistype of ocular tumor. The application of the 651 bp RTEF-1 isoform maybe beneficial in the therapy of other cancers, which rely on VEGFstimulated tumor expansion.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for treating a patient with anangiogenic disorder comprising administering to the patient an effectiveamount of a therapeutic composition comprising a nucleic acid expressionvector comprising a nucleic acid sequence encoding an amino acidsequence at least 95% identical to SEQ ID NO:3, wherein the compositionis administered to the site of the angiogenic disorder and at least onesymptom of the angiogenic disorder is treated.
 2. The method of claim 1,wherein the angiogenic disorder is ocular neovascularization,arterio-venous malformations, coronary restenosis, peripheral vesselrestenosis, glomerulonephritis or rheumatoid arthritis.
 3. The method ofclaim 2, wherein the angiogenic disorder is ocular neovascularization.4. The method of claim 1, wherein the disorder is macular degeneration,corneal graft rejection, corneal neovascularization, retinopathy ofprematurity (ROP) or diabetic retinopathy.
 5. The method of claim 4,wherein the disorder is age-related macular degeneration (AMD).
 6. Themethod of claim 1, further comprising administering a secondantiangiogenic therapy.
 7. The method of claim 6, wherein the secondanti-angiogenic therapy is an antibody that binds to VEGF, a VEGFreceptor, FGF, an FGF receptor, bevacizumab, ranibizumab, or pegaptanibsodium.
 8. The method of claim 1, wherein the angiogenic disorder is acancer.
 9. The method of claim 8, wherein the cancer is a metastaticcancer.
 10. The method of claim 8, wherein the cancer is a bladder,blood, bone, bone marrow, brain, breast, colon, esophagus, eye,gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck,ovary, prostate, skin, stomach, testis, tongue, or uterus cancer. 11.The method of claim 10, wherein the cancer is ocular melanoma.
 12. Themethod of claim 8, further comprising administering a second anticancertherapy.
 13. The method of claim 12, wherein the second anticancertherapy is a chemotherapy, surgical therapy, an immunotherapy or aradiation therapy.
 14. The method of claim 1, wherein the patient is ahuman.
 15. The method of claim 1, wherein the therapeutic composition isadministered to the eye.
 16. The method of claim 15, wherein thetherapeutic composition is administered to the eye topically or byintraocular injection.
 17. The method of claim 16, wherein thetherapeutic composition is administered as an eye drop.
 18. The methodof claim 1, wherein the nucleic acid expression vector is furtherdefined as a viral expression vector.
 19. The method of claim 18,wherein the viral expression vector is an adenovirus, adeno-associatedvirus, herpes virus, SV-40 virus, retrovirus or vaccinia virus vector.20. The method of claim 19, wherein the viral expression vector is anadeno-associated virus.
 21. The method of claim 19, wherein the viralexpression vector is a lentiviral expression vector.
 22. The method ofclaim 21, wherein the lentiviral expression vector is an HIV vector. 23.The method of claim 1, wherein the expression vector comprises a celltype specific or inducible promoter.
 24. The method of claim 23, whereinthe inducible promoter is a hypoxia inducible promoter.
 25. The methodof claim 23, wherein the inducible promoter is an angiogenesis induciblepromoter.